UDC 577.11:611.018.43

Biochemical markers of bone type I metabolism

O. V. Zaitseva, S. G. Shandrenko, M. M. Veliky

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv; e-mail: [email protected]

T his review focuses on the analysis of diagnostic value of the major bone remodeling markers, in particular synthesis and degradation markers of collagen type I. These include carboxy- and aminoterminal telopeptide, carboxy- and aminoterminal propeptide of procollagen type I, hydroxyproline, hydroxylysine, pyridinoline and deoxypyridinoline. Their measurement allows evaluating the structural and functional conditions and also the rate of metabolic processes in the bone tissue. The advantages and disadvantages of determination of these markers in the condition of different bone diseases were examined. It is shown that determination of bone collagen type I metabolism markers is the most informative for assessment of bone resorption, formation and turnover. K e y w o r d s: collagen type I, the markers of collagen synthesis and degradation, bone.

umerous bone diseases, such as osteopo- lytic activity of ALP depends on phosphate concen- rosis, osteomalacia, Paget’s disease, are tration and related to histocytological changes in the N associated with metabolism disorders and bone formation parameters [1, 3]. accompanied by substantial changes in biochemi- Three main ALP isoenzymes have been iden- cal markers of bone remodeling [1]. For diagnosing tified: placental, intestinal and isoenzyme specific and monitoring of bone diseases specific for bone (B-ALP), liver and kidneys. Bone and liver activities, mineral and organic components con- equally contribute to ALP circulation. The intestinal tent in blood are most commonly assessed. These mucosa makes it less. Methods that based on heat components enter the bloodstream in the process of stability, sensitivity to carbamide, peculiarities of their synthesis by osteoblasts and osteoclasts or their electrophoretic mobility, hydrocarbon composition formation in the osteoclasts as a result of mediated and immunochemical specificity are used for the bone resorption. Thus, changes in calcium, inorganic separate determination of individual isoenzymes. In phosphate, osteocalcin, 25-hydroxycholecalciferol particular, bone isoenzyme is thermolabile and B-

(25OHD3) levels [2], alkaline and tartrate-resistant ALP activity is completely inhibited in response to acid phosphatase activities [1], collagen type I me- heat at 56 ºC for 10 min. Residual activity becomes tabolism products such as carboxy- and aminotermi- below 20% of initial activity after heating serum nal propeptides of procollagen type I, hydroxypro- sample that represents partial bone isoenzyme ac- line, hydroxylysine, pyridinoline, deoxypyridinoline tivity [4, 5]. in serum can reflect bone state. ALP activity elevation, mainly due to bone iso- Clinically the measurement of total alkaline , during extensive bone growth and in patho­ phosphatase (ALP) activity and its bone isoen- logic conditions that is accompanied by abnormali- zyme are the most commonly used markers of bone ties in bone mineralization is observed. There are metabo­lism disturbances. ALP is constitutively ex- osteoporosis, osteomalacia, vitamin D deficiency, pressed in osteoblasts (bone forming cells) which aluminium intoxication, hypophosphatemic rickets, catalyzes the phosphate ester hydrolysis during the hyperthyroidism, myocardial infarction, etc. This osteosynthesis and provides phosphate-ion transfer effect can also be associated with long-term use of onto organic components of bone pericellular ma- nonsteroidal anti-inflammatory drugs (allopurinol, trix. ALP is also involved in the process of phos- oral hypoglycemic agents) [3, 5]. phates elevation and their transportation to the site of Osteocalcin (bone Gla-) is a non-col- the hydroxyapatite crystals synthesis in bone. Cata- lagenous bone matrix protein which is prima­rily

ISSN 2409-4943. Ukr. Biochem. J., 2015, Vol. 87, N 1 21 огляди synthesized by mature osteoblasts, odontoblasts calcium-binding TRAP form. That is why it should and hypertrophic chondrocytes. Osteocalcin has be separated before measurement [4, 5, 8]. been found only in bones and in dentin. Three TRAP activity is elevated in patients with γ-carboxyglutamate residues in osteocalcin provide bone metastasis and other diseases accompanied by high Ca2+-ion binding affinity and stabilize the oste- extensive bone destruction, such as osteomalacia, ocalcin α-structure. This allows osteocalcin binding­ hyperthyroidism, Paget’s disease, long-term glu- with the hydroxyapatite. Theoretically, osteocalcin cocorticoid therapy. Like other resorption markers blood level reflects bone formation. However, owing­ TRAP activity is increased after oophorectomy or to post-translational metabolism one-third of osteo- the menopause [5]. calcin is presented in the intact protein form, one- Despite the fact that earlier discussed clinical third in the large N-terminal fragment form and one- markers are widely used in diagnostics, they are not third in the form of medium and small C-terminal universal for the bone conditions assessment. For an fragments. These immunoreactive osteocalcin frag- adequate diagnostics and monitoring of bone dis- ments are released into the blood and can charac­ ease treatment efficacy special attention is paid to terize bone resorption process [6, 7]. the investigation of fragments con- Osteocalcin is a valid bone remodeling marker tent in blood or urine as markers of bone formation in case of coupling between bone formation and re- and resorption. Because collagen is the major bone sorption. When remodeling process is disturbed, os- tissue protein and its amino acid composition differs teocalcin level is tightly related only to osteogenesis. from other in connective tissues, biochemi- It occurs in conditions of long-term use of corticos- cal collagen metabolism markers are specific. These teroids, cancer-associated hypercalcemia, etc. It is markers allow characterizing the bone metabolic known that osteocalcin level is decreased in hypo- processes in normal and pathological conditions. thyroidism, hypoparathyroidism, and myelomatosis. It is well known that various diseases can lead to Osteocalcin in patients with acute lymphoblastic structural changes in collagen. E.g., bone collagen leukemia is decreased two times as compared with in patients with leukemia has different amino acid healthy children [7]. Serum osteocalcin elevation is composition, surface charge, content of covalently observed in primary hyperparathyroidism, hyper- bounded carbohydrate residues and polypeptide thyroidism, Paget’s disease, chronic kidney disease, chain length of molecule α-components compared postmenopausal osteoporosis [5]. However, osteocal­ with normal collagen [9]. The substantial changes in cin is a widely used marker of bone formation but its collagen are specific not only for bone pathologies, short half-life in blood (~10 min) and fast excretion but also for other diseases. This makes collagen an through kidneys should be taked into account [5]. important subject for research. Five acid phosphatase (AP) isoenzymes have Numerous collagen molecule fragments been found. There are bone-, prostate-, platelets-, entering­ the bloodstream during the bone metabolic erythrocyte- and spleen-specific ones. Tartrate-re- lesion have been discovered. Some of them are well sistant acid phosphatase bone isoenzyme (TRAP) studied and widely used in clinical practice, while is produced only by osteoclasts. In this case TRAP the other are potentially important but require a is a specific marker of osteoclasts activity and bone more detailed research. Carboxy- and aminoterminal resorption. Methods for total AP determination and telopeptide, carboxy- and aminoterminal propeptide its isoenzymes activities have low specificity. Never­ procollagen type I, pyridinoline, deoxypyridino- theless, inhibitive capability of TRAP by tartrate line, hydroxiproline and hydroxylisine are the main gives an opportunity to distinguish its activity from biochemical markers of collagen type I metabolism. activities of other phosphatases in particular eryth- The appropriateness of these markers determination rocyte- and prostate-specific isoenzymes. TRAP depends on the peculiarities of pathologies’ develop­ activity measurement is also accompanied by elec- ment, difficulties of laboratory technique and in- trophoretic separation and immunoenzyme analy- formative capacity of obtained data. Therefore, this sis for accuracy improving. The use of TRAP as a review is focused on the analysis of the advantages marker of bone resorption is limited by its instability and disadvantages in clinical using of bone remode­ in frozen serum and plasma samples, as well as by ling markers (resorption and formation) mainly of the presence of an in blood. Addi- collagen type I metabolism markers in various bone tional difficulties are caused by the blood circulating pathologies.

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C ollagen as a major structural helix is more elongated and less twisted. The amino element of bone acid composition of collagen is atypical for proteins. Bone tissue is one of the connective tissue Every third amino acid in the chain is glycine (its types. It performs important functions in the body, content is 33-35%). Collagen is also characterized by such as protection and support for internal organs, high content of proline and hydroxyproline (20-21%). assisting in body movement, reservoir for calcium, These amino acids prevent classic α-helix formation inorganic phosphate and other minerals, the bone and give the collagen chain rigid and bent conforma- marrow also performs blood cell production and tion [12, 17]. functioning of the immune system [10]. Bone has a A major part of the collagen type I complex structure. Its consists (Mr 300 kDa) in mammals (Fig. 1) is synthesized of 35% organic and 65% mineral matrix. Inorganic by fibroblasts and osteoblasts on polyribosomes, matrix is predominantly represented by body cal- which bound to membranes of rough endoplasmic cium (99%), phosphorus (87%), magnesium (~60%), reticulum, as a high-molecular-weight, functiona­ sodium and other elements (25%). Bone calcium lly inactive precursor procollagen type I. The pre- and phosphorus are presented in the hydroxyapatite cursor molecule (Mr 450 kDa) has N-(amino-) and C-(carboxy-) terminal nontriple helix propeptides Ca10(PO4)6(OH)2 mineral form (in the range of 60- 65%) [10, 11]. Organic bone matrix is mainly com- (PINP and PICP) on each of the three chains [19–21]. posed by various collagen types (I, V, VI, XIV and These terminal propeptides are presented in the form XXIV) [12]. Collagen type I is the most abundant of globular or linear domains which play a signifi- among proteins, making up 95% of the total bone cant role in the assembly of the three α-chains into collagen and 80% of the whole bone protein [13, 14]. the triple collagen monomers [22]. Besides collagen type I, there are about 200 non- The newly synthesized procollagen mole­cule is collagenous proteins, such as glycoproteins, proteo- transported to the Golgi apparatus and secreted via glycans, integrin binding proteins, fibronectin, -fi secretory granules into the extracellular space. The bromodulin and enzymes, such as alkaline and acid last stages of the procollagen extracellular modifi- phosphatases which are presented in the bone matrix cation, specifically cleavage of N- and C-terminal [10, 15]. propeptides with forming tropocollagen, take place Bone collagen (type I) is a right-handed helical in the extracellular matrix. This process is catalyzed molecule that consists of three polypeptide chains. by specific Zn2+-dependent N- and C-procollagen peptidase [13, 23]. Formed tropocol- There are two identical α1-chains and one α2-chain lagen can polymerize and aggregate into fibrils to ([α1(I)]2α2(I)) (Fig. 1) [16-18]. The collagen helix structure differs from the form active collagen form. Thus, tropocollagen mon- structure of other α-helical proteins. Thus, collagen omers are assembled along collagen fibrils in colla- helix has 3 amino acid residues per turn unlike 3.6 gen bundles form which are located from each other in α-helix. The helical pitch is 0.9 nm in contrast to at a distance 64 nm. It is considered that these spaces the 0.54 nm in α-helix [12]. So, the primary collagen play a significant role in themineralization. They are

Fig. 1. Molecular structure of fibrillar collagen type I (adapted to [13])

ISSN 2409-4943. Ukr. Biochem. J., 2015, Vol. 87, N 1 23 огляди the initial sites for mine­ral compounds’ deposition. (ICMA). ELISA is preferred in clinical practices for Formed initial crystals are the centers of minerali- measurement these markers in blood or urine (Tab­ zation and hydroxiapatite deposition [12]. During le 1) [26, 27]. collagen fibrils’ formation tropocollagen molecules Level of bone collagen remodeling markers in bound each other by unusual covalent cross-links. blood and urine is affected not only by bone meta- The cross-links’ formation is initiated by oxidation bolic rate, but also by excretion rate of collagen of lysine and hydroxilysine side chains amino groups type I metabolism markers. Thus, we have a ques- to aldehyde groups with allysine and hydroxiallysine tion, which of the markers gives the most reliable formation. These reactions are catalyzed by copper- and accurate characteristic of bone state. A more de- containing enzyme [13, 24, 25]. tailed study of each marker will allow making cer- Owing to extremely large quantities of formed tain conclusions about their experimental and clini- cross-links, bundled and twisted collagen fibrils cal usage [26]. form a 3D network, which is filled up with other substances of the extracellular matrix and makes the Markers of collagen type I degradation tissues sturdier. Collagen posttranslational modifica- 1. Hydroxyproline (HYP) tions are very important for mineralization process. Despite numerous disadvantages, the oldest Thereby, the mineral components deposition occurs and the most commonly used method for collagen only if collagen has a specific structure with availab­ metabolism assessment is HYP level determina- le reactive groups which can act as sites of crystal- tion in the urine. HYP is approximately 13% of all lization [13]. collagen amino acids [28]. HYP synthesis occurs in the condition of collagen mRNA translation on Biochemical markers of bone collagen ribosome. It is catalyzed by prolyl-4-hydroxylase. type I synthesis and degradation This iron-containing enzyme is bound with endo- All skeletal system diseases are accompanied plasmic reticulum membrane. Prolyl hydroxylase has by various bone metabolism disorders. Standard α-ketoglutarate (α-KG) as a co-substrate, which is clinical procedures do not allow in full to charac- decarboxylated and converted to succinate (Fig. 2). terize the actual bone metabolism because most of In hydroxylation process iron of prolyl-4-hydroxy- them are not appropriate for immediate bone assess- lase (Fe2+) gives off electrons and is oxi- ment. Therefore, biochemical markers of collagen dized to Fe3+. Reduction of Fe3+ to Fe2+ is provided type I metabolism determination in dynamics can be by ascorbic acid. determinant for diagnosis or assessment of therapy Proline hydroxylation occurs on the forming effectiveness. Markers of collagen type I metabo- polypeptide chain up to its releasing from the ribo- lism have been classified as shown on Tab­le 1. There some. It is found that attaching a hydroxyl group to are synthesis and degradation markers­. Degrada- proline is necessary for collagen triple helix stabi- tion markers include hydroxyproline (HYP), hydro­ lization and its transportation out of cells. It is also xylysine (HYL), pyridinoline (PYD), deoxypyridi- known that released HYP after collagen breakdown noline (DPD), collagen type I N-terminal telopeptide process cannot be directly reutilized for protein (NTX), collagen type I C-terminal telopeptide synthesis [29]. Hydroxyproline content is related (CTX). Whereas, procollagen type I C-terminal to calcium bloodstream releasing and bone resorp- propeptide (PICP) and procollagen type I N-termi- tion process in various pathologies, such as Paget’s nal propeptide (PINP) are the markers of synthesis. disease, osteoporosis, osteomalacia etc (Calvo et al. These markers reflect changes in bone cells func- 1996) [30]. tioning and show the overall rate of bone collagen The most commonly used method for HYP metabolism in pathology [26]. Synthesis and degra- specific determination in clinics is based on HYP dation markers differ in their specificity and ability oxidation by hydrogen peroxide in alkaline medium to detect. Various methods can be used to identify with the Cu2+ presence. Addition of p-dimethylami­ and measure their levels. There are radio immuno nobenzaldehyde after acidification leads to genera- assay (RIA), enzyme-linked immunosor­bent assay tion of the pink color [31]. (ELISA), high performance liquid chromatography HYP exists in biological fluids in various (HPLC), electrochemiluminescence immunoas- forms. Approximately 90% of HYP which released say (ECLIA), immunochemiluminometric assay during collagen breakdown (especially during bone

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Ta b l e 1. Synthesis and degradation markers of collagen type I

Biological Analytical M arker and its specificity Tissue origin samples method Markers of collagen degradation Hydroxyproline (HYP); specific for all fibrillar Bone, skin, Urine Colorimetry, and collagen proteins, including ; found in newly cartilage, HPLC synthesized and mature collagen soft tissues Hydroxylisine-glycosides (Hyl-Glyc); specific for Bone, skin, Urine HPLC, collagens and collagen proteins; glucogalactosyl- soft tissues ELISA hydroxilysine is highly contained in soft tissue collagens and C1q; galactosyl-ОНLys is highly contained in skeletal collagen Pyridinoline (PYD, Pyr); high concentration is observed Bone, tendon, Urine, HPLC, in cartilage and bone collagen; not found in skin, found cartilage, serum ELISA only in mature collagen blood vessels Deoxypyridinoline (DPD, d-Pyr); high concentration Bone, dentine Urine HPLC, is observed only in bone tissue collagen; not found in ELISA cartilage and skin; found only in mature collagen Collagen type I N-terminal telopeptide (NTX); high levels All tissues Urine, ELISA, RIA, are found in bone collagen type I which contain serum ECLIA collagen type I Collagen type I C-terminal telopeptide All tissues Urine, ELISA, RIA, (α-CTX fragments, β-CTX); high levels which contain serum ECLIA are found in bone collagen type I collagen type I Markers of collagen synthesis C-terminal propeptide procollagen type I (PICP) Bone, skin, Serum RIA, ELISA soft tissues N-terminal propeptide procollagen type I (PINP) Bone, skin Serum RIA, ELISA

Fig. 2. Scheme of proline hydroxylation by prolyl hydroxylase

ISSN 2409-4943. Ukr. Biochem. J., 2015, Vol. 87, N 1 25 огляди resorption) circulates in plasma in the free amino containing collagen-like sequences. Lysine hydroxy- acid form. After filtration and almost complete renal lation occurs in the presence of . reabsorption, HYP is oxidized in the liver yielding This process is necessary for the covalent bonds urea and carbon dioxide. Approximately 10% of re- formation between collagen molecules in the fibrils’ leased HYP circulates in blood in the protein-bound formation. The hydrocarbon residues, like galactose form. These proteins are filtered and excreted into and galactosyl-glucose disaccharide, attach to the the urine without metabolic alterations [32, 33]. procollagen molecule after hydroxylation process. It is considered that collagen is one source of It is catalyzed by specific glycosyltransferases. As a HYP in the body. Recently it was discovered that result HYL is converted to galactosyl-hydroxylisine other animal proteins such as elastine, C1q, acetyl and glicosyl-galactosyl-hydroxylysine (Fig. 3) [12]. cholinesterase, glycoproteins obtained by lung lava­ The relative and total content of galactosyl-hy- ge also contain HYP [34]. Therefore, in case of HYP droxylysine and glycosyl-galactosyl-hydroxylysine urine measurement the HYP contribution, released in bone and soft tissues are different. It allows sug- during non-collagenous proteins metabolism and gesting that the urine glycosides’ level is more sensi- procollagen type I N-terminal propeptide (PINP) tive marker of bone resorption and collagen molecule should be considered. It also should be noted that degradation as compared with the urine HYP [37]. HYP obtained from PINP images more bone forma- Released HYL and HYP are not involved in tion than bone resorption [35]. Inflammation [36] collagen biosynthesis. Although HYL content in and dietary collagen [35] can also lead to a signifi- collagen is less than HYP content, but HYL is a cant HYP level increasing. Thus, total HYP urine prospective marker of this protein degradation. This level partly reflects collagen degradation. marker needs further research [37, 38]. Because of HYP availability in different pro- 3. Pyridinoline (PYD) and deoxypyridinoline teins and due to its metabolism peculiarities the (DPD) HYP level is weakly related with the actual bone Deamination of lysine or hydroxylysine collagen metabolism. All of these data indicate the ε-amino group in the presence of lysyl oxidase leads necessity of using more sensitive and specific col- to the cross-links formation with 3-hydroxypyridine lagen type I degradation markers. ring in their structure (Fig. 4) [39, 27]. In human 2. Hydroxylysine (HYL) urine samples two types of cross-links are identi- HYL is another modified amino acid that deter- fied. There are DPD and YP D. These cross-links are mines the unique collagen composition and proteins formed during extracellular maturation of the fibril-

Fig. 3. Carbohydrate components of collagen

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Pyridinoline Deoxypyridinoline

Fig. 4. Pyridine cross-links of collagen type I lar collagens. Interaction between two hydrolysine (60%) [42]. DPD and PYD excretion is controlled and one lysine residues leads to the deoxypyridino- by circadian rhythm: the highest excretion occurs at line formation. Pyridinoline cross-links are formed night and the lowest – in the day time [43]. Some as a result of interaction between three hydrolysine other factors can also affect the PYD and DPD level residues of collagen molecule. in the urine. It may increase 2 times during pregnan- These cross-links stabilize the collagen struc- cy. The urine DPD excretion for females in meno­ ture. PYD and DPD ratio in human bone is about pause is 2-3 times higher than for females in fer- 3:4. PYD and DPD are formed during the post- tile age. Diet and physical activity have no effect on translational collagen modification which have been DPD and PYD excretion. Urine DPD level is higher already secreted and incorporated into the extracel- for females than for males. For both sexes PYD and lular matrix. That is why they cannot be directly reu- DPD content in the urine increases in cases of pri- tilized for collagen synthesis [39]. mary hyperparathyroidism (about 3 times) [43], hy- High pyridinoline concentration is observed perthyroidism (about 5 times) and Paget’s disease (12 in the collagen type II of cartilage. Ppyridinoline times). Also significant increasing of DPD excretion has also been found in the bone collagen. The main is observed in cases of osteoporosis, osteoarthritis deoxypyridinoline source is bone and dentin. Both and rheumatoid arthritis [44]. DPD and PYD can be found in collagen tendon and The determination of pyridine cross-links level aorta, but are not detected in the skin [40]. Because in the urine has several potential advantages. There the major organic component of bone is collagen are high specificity of pyridine cross-links for bone matrix, which has a rapid metabolic rate, it can be turnover, absence of DPD and PYD metabolic trans- assumed that bone is the main source of PYD and formation in vivo before the urine excretion and their DPD in biological fluids. Because DPD and PYD are determination without any prior dietary limitations formed during collagen molecule maturation they [45]. were considered as specific markers of bone resorp- The urine DPD and PYD content can be de- tion and collagen type I degradation [41]. termined by the HPLC analysis, ELISA fluoromet- PYD and DPD enter the bloodstream during ric and chemiluminescent methods. In particular, bone collagen matrix proteolytic degradation. As ELISA is the most commonly used method that al- low-molecular-weight compounds, they are excreted ­ lows determining free DPD and PYD form. Howe­ with urine in free (40%) or peptide-bound form ver, these results should be accompanied by other

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N-telopeptide C-telopeptide

Fig. 5. Localization of N- and C-telopeptide in the collagen type I diagnostic tests, because the DPD and PYD content Markers of type I collagen formation in the urine does not fully characterize normal or 1. Procollagen type I terminal propeptides pathological bone state [19, 45]. (PINP, PICP) 4. Collagen type I telopeptides (CTX, NTX) During extracellular collagen type I maturation Fragments of carboxy- (CTX) and amino- (before fibrils’ formation) the N-terminal (PINP) (NTX) terminal collagen cross-links are released in and C-terminal (PICP) propeptides are removed the bloodstream as a result of osteoclast-mediated from pro­collagen molecule by specific endopepti- collagen type I degradation. It occurs under bone re- dase. Pro­collagen molecule is by 50% longer than sorption by action of osteoclasts proteolytic enzyme mature collagen. Propeptides removing is necessary cathepsin K. As compounds with small size, they are readily pass through the glomerulus and excreted for early collagen molecule aggregation into fibrils with urine (Fig. 5) [19]. inside the cells. NTX fragments can be identified in the serum Procollagen type I C-terminal propeptide is a glycoprotein (M 115 kDa) which is composed of and urine. NTX level is significantly decreased af- r ter the anti-resorptive therapy. It is currently unclear three polypeptide chains. There are two α1-I poly- the influence of diet on the NTX level. Monoclonal peptide chains, containing 246 amino acid residues antibodies for NTX determination that specifically and one α2-I polypeptide chain, containing 247 recognize the α-2 chain N-telopeptide fragment are amino acid residues. Each of these chains contains used in clinics [19]. CTX fragments in the urine also oligosaccharides with high mannose content. Intra- can be determined by ELISA. and intermolecular disulfide bonds aggregate subu- After racemization and isomerization α-CTX nits with a globular structure formation (Table 2) can form to β-CTX (α-aspartic acid is converted to [26]. This structure is necessary for the normal triple β-form). That is why monoclonal antibodies which structure formation of mature collagen type I mole­ can specifically recognize α- or β-CTX are used cules. [46]. α-CTX indicates the breakdown of the newly- Due to the high mannose content, PICP is me- synthesized collagen while ß-CTX of the mature col- tabolized in the liver endothelial cells by means of lagen. Based on these data it is proposed to calculate the mannose-6-P receptors. PICP half-life is about the α-CTX/ß-CTX ratio and use obtained index as a 6-8 minutes [49, 50]. value of bone metabolism rate. E.g., α-CTX/ß-CTX Procollagen type I N-terminal propeptide index is increased in patients with Paget’s disease (Mr 70 kDa) is a heterotrimer without covalent and rapidly decreased under the biophosphonates bounds in its triple structure. PINP is composed influence. This index in serum is increased almost of two α1-I polypeptide chains and one α2-I chain. 2 times during menopause [46]. These chains are held together by identical to the Because osteoclasts affect only bone collagen collagen molecule structure short helical sites. These type I CTX and NTX fragments are the most informa­ collagen-like sites define PINP properties. Thus, tive markers of bone collagen destruction [47, 48]. PINP antigenicity is increased at its interaction with Thus, determination of CTX and NTX level in dy- a small helical domain of α1-I chain Col-1, which is namics is used for bone diseases diagnosing and bone the most immunogenic part of PINP propeptide [50]. resorption monitoring during antiresorptive therapy. PINP is metabolized by livers’ macrophages and its These markers allow evaluating the efficacy of bone blood level is 100 times higher than the PICP con- diseases therapy after 3 months of treatment [26]. centration [44, 50].

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Ta b l e 2. Comparative characteristics of procollagen type I propeptides

Propeptides PINP PICP Localization aminoterminal carboxyterminal Molecular weight 70 kDa 115 kDa Structure linear globular Chemical nature phosphoprotein glycoprotein Bonds non-covalent disulfide

The maturation of soft tissue collagen type I Periostin is a Gla-containing extracellular ma- can contribute to the total level of PINP and PICP. trix protein. Periostin is located in the periosteum However, this contribution has the minimum value­, and affects the regulation of bone formation and because the main part of PINP and PICP is released its strength through the PYD and DPD cross-links during bone collagen type I formation. The ratio be- formation in collagen [52]. Sklerostin stimulates tween collagen, which is deposited in bone matrix secretion of RANK-L which is formed in osteo- and PICP (or PINP), which is released into the blood- cytes. Sklerostin also enhances osteoclasts’ activi­ stream, are theoretically equal to 1. Thus, based on ty. Circulating factor FGF-23 which is expressed in blood propeptides’ level, it is possible to characteri­ osteocytes, negatively affects the serum levels of ze the ability of osteoblasts to synthesize collagen inorganic phosphate and 1,25-dihydroxyvitamin D3 type I. As propeptides with high molecular weight, [53]. MicroRNAs play a significant role in normal PINP and PICP cannot be filtered through the kid- differentiation and functioning of the bone cells [54]. ney. So, their level is not affected by kidney filtration Thus, many new biomarkers of bone tissue re- [27]. modeling have been recently discovered and inves- High level of PICP and PINP is observed in tigated. Their use along with well-studied markers pathologies associating with increased bone re- will allow detecting and preventing numerous bone modeling processes in particular Paget's disease, diseases. hyperthyroidism, primary hyperparathyroidism and neurogenic osteodystrophy. In some cases, ele­ Біохімічні маркери vated PICP level is observed in early menopause. метаболізму колагену І типу Low PICP concentrations are found in children with кісткової тканини growth hormone deficiency [51]. О. В. Зайцева, С. Г. Шандренко, Data from numerous investigations indicate М. М. Великий that PINP and PICP reflect bone tissue formation. That is why PINP and PICP are significant markers Інститут біохімії ім. О. В. Палладіна for monitoring bone diseases [26, 51]. НАН України, Київ; Biochemical markers of bone synthesis and e-mail: [email protected] degradation are an important tool for assessing and monitoring skeletal system conditions. As compared В огляді проведено аналіз діагностичного with standard clinical methods, determination of the значення основних маркерів ремоделюван- potential new bone metabolism markers allows in- ня кісткової тканини, зокрема, синтезу та vestigating the rate of bone metabolism and sponta- деградації колагену I типу, до яких належать neous bone loss, performing the bone diseases moni- карбокси- та амінотермінальні телопептиди, toring and prognosis of the risk of fractures. Besides карбокси- та амінотермінальні пропептиди про- the main markers of mineral metabolism, synthesis колагену I типу, гідроксипролін, гідроксилізин, and degradation of collagen type I (CTX, NTX, піридинолін та дезоксипіридинолін. Їх виз- PINP, PICP, GP, GL, DPD, PYD), new markers­ of начення дозволяє оцінити структурно- bone remodeling have been discovered. These in- функціональний стан та швидкість обмінних clude periostin (POSTN), osteocytes’ factors: scle- процесів у кістковій тканині. Розглянуто перева- rostin and FGF-23, circulating in blood microRNA ги та недоліки методів визначення цих маркерів (miRNA) [44]. за різних кісткових захворювань. Показано, що

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визначення маркерів метаболізму колагену I 3. komisarenko S. V., Apukhovska L. I., Rias­ типу кісткової тканини є найінформативнішим niy v. m., Kalashnikov A. V., Veliky N. N. для оцінки кісткової резорбції, формування та “Mebivid” biopharmaceutical preparation

ремоделювання. efficacy against vitamin3 D and calcium metabolism disorders in alimentary К л ю ч о в і с л о в а: колаген I типу, марке- osteoporosis. Biotechnol. Acta. 2011;4(1):74-81. ри синтезу та деградації колагену, кісткова тка- (In Ukrainian). нина. 4. Blackwell P., Godber I. M., Lawson N. Biochemical markers of bone turnover. – In: Биохимические маркеры Clinical Trials in Osteoporosis (Second Edition) / метаболизма коллагена D. Pearson, C. J. Miller (Eds.). London: Springer- І типа костной ткани Verlag, 2007:247-269. О. В. Зайцева, С. Г. Шандренко, 5. Riggs B. L., Melton L. J. Osteoporosis etiology, Н. Н. Великий diagnosis, and management. M.: Binom, 2000. 560 p. И нститут биохимии им. А. В. Палладина 6. Lee J. A., Hodges S., Eastell R. Measurment of НАН Украины, Киев; osteocalcin. Ann. Clin. Biochem. 2000;37:432- e-mail: [email protected] 446. В обзоре проведен анализ диагностиче- 7. Bebeshko V. G., Bruslova K. M., Panchenko L. M., ского значения основных маркеров ремодели- Volodina T. T., Astachova V. S., Stvetkova N. M., рования костной ткани, в частности, синтеза и Mishchenko L. P., Trychlib I. V. Influence of bone деградации коллагена I типа. К ним относятся marrow colony forming fibroblasts units (CFFU) карбокси- и аминотерминальные телопептиды, efficiency on disease-free life expectancy for карбокси- и аминотерминальные пропептиды children with acute lymphoblastic leukemia. проколлагена I типа, гидроксипролин, гидрок- Ukr. J. Hematol. Blood Transfus. 2010;10(6):5-7. силизин, пиридинолин и дезоксипиридинолин. (In Ukrainian). Их определение позволяет оценить структурно- 8. Civitelli R. Biochemical markers of bone функциональное состояние и скорость обмен- turnover. In: The Osteoporotic Syndrome. ных процессов в костной ткани. Рассмотрены Detection, prevention, and treatment (Fourth преимущества и недостатки методов определе- Edition) / L. V. Avioli (Ed.). New York: Academic ния этих маркеров при различных костных за- Press, 2000. P. 67-89. болеваниях. Показано, что определение марке- 9. volodina T. T., Pechenova T. M., Dzvon­ ров метаболизма коллагена I типа костной ткани kevich N. D., Popova N. N., Silonova N. V., является наиболее информативным для оценки Astahova V. S., Panchenko L. M., Guly M. F., костной резорбции, формирования и ремодели- Mikhaylovsky V. O. Modifying effect of рования. low-molecular metabolities on the state of extracellular matrix of connective tissue of К л ю ч е в ы е с л о в а: коллаген I типа, animals. Ukr. Biokhim. Zhurn. 2007;79(6):65-73. маркеры синтеза и деградации коллагена, кост- (In Ukrainian). ная ткань. 10. Kini U., Nandeesh B.N. Physiology of bone formation, remodeling, and metabolism. References Radionuclide and Hybrid Bone Imagine. 1. Gayko G. V., Kalashnikov An. V., Brusko A. T., 2012;XIV:29-57. Apukhovska L. I., Kalashnikov Al. V. Vita­ 11. Jayachandran V., Se-Kwon K. Chitosan mine D and bone system. Kyiv: Book plus, 2008. composites for bone tissue engineering – An 176 p. (In Russian). Overview. Mar. Drugs. 2010;8(8):2252-2266. 2. Veliky M. M., Zaytseva O. V., Shymanskyy I. O., 12. Shoulders M. D., Raines R. T. Collagen structure Shandrenko S. G., Latyshko N. V., Gudkova O. O., and stability. Ann. Rev. Biochem. 2009;78:959- Mazanova A. O., Apukhovska L. I. The features 958. of biomolecule peroxidation processes in rat 13. Gelse K., Poschl E., Aigner T. Collagens –

liver under conditions of vitamin D3 deprivation. structure, function, and biosynthesis. Adv. Drug Biol. Systems. 2013;5(3):287-294. (In Ukrainian). Deliv. Rev. 2003;55:1531-1546.

30 ISSN 2409-4943. Ukr. Biochem. J., 2015, Vol. 87, N 1 o. v. zaitseva, s. g. shandrenko, m. m. veliky

14. Viguet-Carrin S., Garnero P., Delmas P. D. The fibroblasts by regulating levels of procollagen role of collagen in bone strength. Osteoporos. C-proteinase. Exp. Cell Res. 1999;252(2):319- Int. 2006:17:319-336. 331. 15. Trammel L. H., Kroman A. M. Bone and dental 26. Čepelak I., Čvorišćec D. Biochemical markers of histology. In: Research Methods in Human bone remodeling – review. Biochemia Medica. Skeletal Biology / E. A. Di Gangi, M. K. Moore 2009;19( 1):17-35. (Eds.). Elsevier Inc., 2013. P. 361-395. 27. Seibel M. J. Biochemical markers of bone 16. Kramer R. Z., Bella J., Brodsky B., Berman H. M. turnover part I: Biochemistry and Variability. The crystal and molecular structure of a Clin. Biochem. Rev. 2005;26(4):97-122. collagen-like peptide with a biologically relevant 28. Shiqemura Y., Kubomura D., Sato Y., Sato K. sequence. J. Mol. Biol. 2001;311(1):131-147. Dose-dependent changes in the levels of free 17. Knupp C., Squire J. M. Molecular packing and peptide forms of hydroxyproline in human in network-forming collagens. Sci. World J. plasma after collagen hydrolysate ingestion. 2003;3:558-577. Food Chem. 2014;159(15):328-332. 18. Saeidi N., Karmelek K. P., Ruberty J. A. 29. Gorres K. L., Raines R. T. Prolyl 4-hydroxylase. Molecular crowding of collagen: A pathway Crit. Rev. Biochem. Mol. Biol. 2010;45(2):106- to produce highly-organized collagenous 124. structures. Biomaterials. 2012;33(30):7366-7374. 30. Babeshko V. G., Bruslova K. M., Kusher O. V., 19. Shaw N., Högler W. Biochemical markers of Babeshko O. V., Volodina T. T. Assessment bone metabolism. In: Pediatric Bone (2nd Ed.) / of bone tissue condition under the amino acid H. F. Glorieux, J. M. Pettifor, H. Juppner (Eds.). composition of urine. Ukr. J. Hematol. Blood 2012. P. 361-381. Transfus. 2009;(2):24-26. (In Ukrainian). 31. Ignat’eva N. Yu., Danilov N. A., Averkiev S. V., 20. Paolino D., Cosco D., Cilurzo F., Trapasso E., Obrezkova M. V., Lunin V. V., Sobol E. N. Morittu V. M., Celia C., Fresta M. Improved Determination of hydroxyproline in tissues and in vitro and in vivo collagen biosynthesis by the evaluation of the tissues. J. Anal. Chemistry. asiaticoside-loaded ultradeformable vesicles. 2007;62(1):51-57. J. Control. Release. 2012;162(1):143-151. 32. Jagtap V. R., Ganu J. V. Effect of antiresorptive 21. Ono S., Imai T., Shimizu N., Nakayama M., therapy on urinary hydroxyproline in Yamano T., Tsumura M. Serum markers of postmenopausal osteoporosis. Indian J. Clin. type I collagen synthesis and degradation in Biochem. 2012;27(1):90-93. amyotrophic lateral sclerosis. Eur. Neurol. 33. Atlante A., Passarella S., Quagliariello E. 2000;44(1):49-56. Spectroscopic study of hydroxyproline transport 22. Ricarrd-Blum S., Ruqqiero F. The collagen in rat kidney mitochondria. Biochem. Biophys. superfamily: from the extracellular matrix to Res. Commun. 1994;202(1):58-64. the cell membrane. Pathol. Biol. 2005;53(7):430- 34. Volodina T. T., Pechenova T. M., Petrun L. M., 442. Alesina M. U. Condition of bone connective 23. Prokop D. J., Sieron A. L., Li S. W. Procollagen tissue matrix and coating tissue of rats under N – proteinase and procollagen C-proteinase. combined exposure. Kyiv: Atika, 2006. P. 145- Two unusual metaloproteinases that are 156. (In Ukrainian). essential for procollagen processing probably 35. Hess B. J., Johnston M. M., Iobst W. F., have important roles in development and cell Lipner R. S. Practice-based learning can signaling. Matrix Biol. 1998;16(7):399-408. improve osteoporosis care. J. Am. Geriatr. Soc. 24. Jensen C. H., Hansen M., Brandt J., Rasmus­ 2013;61(10):1651-1660. sen H. B., Jensen P. B., Teisner B. Quantification of 36. Millet P., Vilaseca M.A., Valls C., Pérez- the N-terminal propeptide of human procollagen Dueñas B., Artuch R., Gomez L., Lambru­ type I (PINP): Comparison of ELISA and RIA scihini N., Campistol J. Is deoxypyridinoline with respect to different molecular forms. Clin. a good resorption marker to detect osteopenia Chim. Acta. 1998;269(1):31-41. in phenylketonuria? Clin. Biochem. 25. Parsons M., Kessler E., Laurent G. J., 2005;38(12):1127-1132. Brown R. A., Bishop J. E. Mechanical load 37. Lo Cascio V., Bertoldo F., Gambaro G., enhances procollagen processing in dermal Gasperi E., Furlan F., Colapietro F., Lo

ISSN 2409-4943. Ukr. Biochem. J., 2015, Vol. 87, N 1 31 огляди

Cascio C., Campaqnola M. Urinary galactosyl- 46. Civitelli R., Armamento-Villareal R., Napoli N. hydroxylysine in postmenopausal osteoporotic Bone turnover markers: understanding their women: A potential marker of bone fragility. value in clinical trials and clinical practice. J. Bone Miner. Res. 1999;14(8):1420-1424. Osteoporos. Int. 2009;20(6):843-851. 38. Langrock T., Garcia-Villar N., Hoffman R. 47. Tanzy M. E., Camacho P. M. Effect of Analysis of hydroxyproline isomers and vitamin D therapy on bone turnover markers in hydroxylysine by reversed-phase HPLC and mass postmenopausal women with osteoporosis and spectrometry. J. Chromatography. B, Analyt. osteopenia. Endocr. Pract. 2011;17(6):873-879. Technol. Biomed. Life Sci. 2007;847(2):282-288. 48. Baim S., Miller P.D. Assessing the clinical utility 39. Eriksen H. A., Sharp C. A., Robins S. P., of serum CTX in postmenopausal osteoporosis Sassi M. L., Risteli L., Risteli J. Differently and its use in predicting risk of osteonecrosis of cross-linked and uncross-linked carboxy- the jaw. J. Bone Miner. Res. 2009;24(4):561-574. terminal telopeptides of type I collagen in human 49. Smedsrod B., Melkko J., Restelli L., Restelli J. mineralised bone. Bone. 2004;34(4):720-727. Circulating C-terminal propeptide of type I 40. Stevenson D. A., Schwarz E. L., Viskochil D. H., procollagen is cleared mainly via the mannose Moyer-Mileur L. J., Murray M., Firth S. D., receptor in liver endothelial cells. Biochem. J. D’Astous J. L., Carey J. C., Pasquali M. Evidence 1990;271(2):345-350. of increased bone resorption in neurofibromatosis 50. Risteli J., Niemi S., Kauppila S., Melkko J., type 1 using urinary pyridinium crosslink Risteli L. Collagen propeptides as indicators analysis. Pediatr. Res. 2008;63(6):697-701. of collagen assembly. Acta Orthop. Scand. 41. Seibel M., Woitge W. Basic principles and 1995;66(266):183-188. clinical applications of biochemical markers of 51. Leeming D. J., Koizumi M., Qvist P., Barkholt V., bone metabolism: Biochemical and technical Zhang C., Henriksen K., Byrjalsen I., aspects. J. Clin. Densitom. 1999;2(3):299-321. Karsdal M. A. Serum N-terminal propeptide of 42. Vesper H. W., Audain C., Woolfitt A.,O spina M., collagen type I is associated with the number of Barr J., Robins S. P., Mayers G. L. High- bone metastases in breast and prostate cancer performance liquid chromatography method and correlates to other bone related markers. to analyze free and total urinary pyridinoline Biomark Cancer. 2011;3:15-23. and deoxypyridinoline. Anal. Biochem. 52. Li X., Zhang Y., Kang H., Liu W., Liu P., Zhang J., 2003;318(2):204-211. Harris S. E., Wu D. Sclerostin binds to LRP5/6 43. Fassbender W. J., Gödde M., Brandenburg V. M., and antagonizes canonical Wnt signaling. Usadel K. H., Stumpf U. C. Urinary bone J. Biol. Chem. 2005;280(20):19883-19887. resorption markers (deoxypyridinoline and 53. Shimada T., Hasegawa H., Yamazaki Y., C-terminal telopeptide of type I collagen) in Muto T., Hino R., Takeuchi Y., Fujita T., healthy persons, postmenopausal osteoporosis Narahara K., Fukumoto S., Yamashita T. FGF- and patients with type I diabetes. Adv. Med. Sci. 23 is a potent regulator of vitamin D metabolism 2009;54(1):1-6. and phosphate homeostasis. J. Bone Miner. Res. 44. Garnero P. New developments in biological 2004;19(3):429-435. markers of bone metabolism in osteoporosis. 54. Lian J. B., Stein J. S., van Wijnen A.J., Stein J. L., Bone. 2014;(66):46-55. Hassan M. Q., Gaur T., Zhanq Y. Micro RNA 45. Abe Y., Ishikawa H., Fukao A. Higher efficacy of control of bone formation and homeostasis. Nat. urinary bone resorption marker measurements in Rev. Endocrinol. 2012;8(4):212-227. assessing response to treatment for osteoporosis in postmenopausal women. Tohoku J. Exp. Med. Received 15.07.2014 2008;214(1):51-59.

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